Biomarker and composition for diagnosis of preeclampsia

ABSTRACT

The present invention relates to a biomarker and a composition for diagnosis of preeclampsia. In accordance with one aspect of the present invention, there is provided a biomarker for diagnosis of preeclampsia using an enzyme selected from the group consisting of placental chondroitin 4-O-sulfotransferase 1 (C4ST), chondroitin 6-sulfotransferase (C6S), heparan sulfate 6-O-sulfotransferase 1 (HS6S), and dermatan/chondroitin sulfate 2-sulfotransferase (CS-2OST), or uronic acid-2-sulfate (UA2S).

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to and the benefit of Korean PatentApplication No. 10-2007-0069002 filed in the Korean IntellectualProperty Office on Jul. 10, 2007, the entire content of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a biomarker and a composition fordiagnosis of preeclampsia.

BACKGROUND OF THE INVENTION

Preeclampsia in pregnancy can be a very serious health problem. It cancause fetal growth restriction, fetal death and morbidity, prematuredeliveries, and death of the mother. The exact cause of preeclampsia isnot known, and treatments for efficiently curing or preventingpreeclampsia are not also available yet. Preeclampsia is known to causeseveral problems at the same time, such as high blood pressure(hypertension), pathological edema and leakage of protein into the urine(proteinuria). Further, preeclampsia is one of the pregnancycomplications that bring hypertension, proteinuria and traumatism to themother. It is known that preeclampsia occurs to only about 3-5% ofpregnant women, but it can seriously affect both the mother and herunborn (or newborn) baby, and thus, acts as a major cause of increasingperinatal mortality and morbidity rates.

Globally, at least 200,000 pregnant women die from preeclampsia everyyear. Its symptoms typically become evident after the 20^(th) week ofpregnancy. Preeclampsia is usually diagnosed by detecting high bloodpressure of a pregnant woman or by checking her urine for protein. Earlydiagnosis and timely treatment of preeclampsia can remarkably reducerisks to the mother and her unborn baby, but such a monitoring method byusing those symptoms as criteria is not effective for an early diagnosisof preeclampsia. Further, no treatments are currently available to curepreeclampsia. Preeclampsia can be mild, but potentially life-threateningdepending on the severity of the disease. Despite such clinical risks,however, it is difficult to find the cause or the pathogenesis ofpreeclampsia at an early stage, or to make an early diagnosis andprognosis.

Therefore, if it becomes possible to suggest the pathogenesis ofpreeclampsia and make an early diagnosis and prognosis based on thesame, the mother having preeclampsia and her unborn baby can beprotected, and the death rate would be reduced. Even if many researcheshave been conducted to monitor and predict the occurrence ofpreeclampsia, they are limited to using a specific protein or substance,which is not sufficient to explain the whole phenomenon about theoccurrence of preeclampsia and the pathogenesis thereof.

While the inventors of the present invention are trying to discover thepathogenesis of preeclampsia through the comparison between normalpregnant women and patients with preeclampsia (whether mild or severe),they have learned that the patients with preeclampsia express loweramounts of diverse glycosaminoglycans (GAGs) in the placenta andplacenta enzymes responsible for sulfonation of GAGs. Based on this, theinventors developed a new way for the early diagnosis and prognosis ofpreeclampsia by preparing a biomarker and a composition.

SUMMARY OF THE INVENTION

It is, therefore, a primary object of the present invention to provide anovel biomarker to detect preeclampsia developed in the placenta of apregnant woman.

It is another object of the present invention to provide a compositionfor the diagnosis of preeclampsia based on the amounts of diverse GAGsin the placenta and placenta enzymes responsible for sulfonation ofGAGs.

It is still another object of the present invention to provide apredictive biomarker kit capable of predicting a risk of preeclampsia.

In accordance with one aspect of the present invention, there isprovided a biomarker for diagnosis of preeclampsia using an enzymeselected from the group consisting of placental chondroitin4-O-sulfotransferase 1 (C4ST), chondroitin 6-sulfotransferase (C6S),heparan sulfate 6-O-sulfotransferase 1 (HS6S), and dermatan/chondroitinsulfate 2-sulfotransferase (CS-20ST), or uronic acid-2-sulfate (UA2S).

In accordance with another aspect of the present invention, there isprovided a composition for diagnosis of preeclampsia, comprising one ormore primer pairs selected from the group consisting of primer pairswith the sequences of: (Primer Pair No. 1) Forward:5′GTGGGGAGAGGGAGAGAATC3′ (Sequence No. 1), Reverse:5′ACAGACAAGAACGACCCATC3′ (Sequence No. 2); (Primer Pair No. 2) Forward:5′CCCAAAGTCAGAAAGCGAAG3′ (Sequence No. 3), Reverse:5′ACAAGCAAACCCACCAACTC3′ (Sequence No. 4); (Primer Pair No. 3) Forward:5′TCTGAGCCTGACCACAGATG3′ (Sequence No. 5), Reverse:5′CACCTGCACAGAACTCAGGA3′ (Sequence No. 6); (Primer Pair No. 4) Forward:5′CCCAGTGGCCCTAAAGTACA3′ (Sequence No. 7), Reverse:5′GTCCATCACTTTGGCAGGTT3′ (Sequence No. 8); and (Primer Pair No. 5)Forward: 5′GTACAACCTGGCCAACAACC3′ (Sequence No. 9), Reverse:5′CGCGTGCTATTGTACTGCAT3′ (Sequence No. 10).

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The above and other objects and features of the present invention willbecome apparent from the following description of the preferred examplesgiven in conjunction with the accompanying drawings, in which:

FIG. 1 shows a polyacrylamide gel electrophoresis (PAGE) assay pictureof a GAG isolated from other tissue (where lane std denotes a heparinoligosaccharide standard, lanes 1 to 12 correspond to GAGs from sample#1 to #12 (refer to Table 2), and lane HS denotes a heparan sulfatecontrol group);

FIG. 2 represents a chromatographic result of CS (chondroitin sulfates)disaccharide standard; and

FIG. 3 presents a chromatographic result of Hep/HS disaccharidestandard.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the following detailed description, reference is made to theaccompanying drawings and sequence listing that show, by way ofillustration, specific embodiments in which the invention may bepracticed. These embodiments are described in sufficient detail toenable those skilled in the art to practice the invention. It is to beunderstood that the various embodiments of the invention, althoughdifferent, are not necessarily mutually exclusive. The followingdetailed description is, therefore, not to be taken in a limiting sense,and the scope of the present invention is defined only by the appendedclaims that should be appropriately interpreted along with the fullrange of equivalents to which the claims are entitled.

Then, experiments performed for better understanding the presentinvention will be described in detail as follows, which are set forth toillustrate, but are not to be construed to limit the present invention.

The inventors confirmed by using RT-PCR that patients who developedpreeclampsia expressed a lower amount of placenta enzymes, which areinvolved in sulfonation of GAGs. GAGs cover plasma membranes of allcells and fill extracellular matrix composed of heparin sulfate (HS),CS, and dermatan sulfate (DS). The inventors confirmed that mRNA levelsof C4ST, C6S, HS6S and CS-2OST were reduced in patients withpreeclampsia. Particularly, the inventors turned out that patients withpreeclampsia did not have other isoforms of the last enzyme (i.e.,CS-2OST), which cause C2-sulfonation on uronic acid or iduronic acid inCS with disaccharide repeat units. This decrease in the amount of theenzyme was confirmed through the lack of UA2S in a placental sampletested for preeclampsia.

Further, the inventors learned from RT-PCR assay results that the amountof CS-2OST was noticeably decreased in the placenta of a woman affectedby preeclampsia (this was also the same for other GAGsulfotransferases). The inventors also confirmed through thedisaccharide assay that UA2S (the final product of sulfate2-sulfotransferase), which is the disaccharide repeat unit of placentalGAGs, was completely disappeared from a disease-infected sample.

The inventors assumed that preeclampsia might be associated with theprogress of glycomics of the placental abnormalities. This assumptionwas confirmed by monitoring decrease in the placental mRNA expression ofdiverse GAG sulfotransferases in preeclamptic pregnancy.

One of the important findings through the analysis of CS disaccharidecomposition for this invention is that preeclamptic pregnancies lack theexpression of UA2S, and this supports parallel decrease observed in theexpression of CS-2OST. This is very clear in that the enzyme has only asingle heteroplasm in most entities with known genome sequences, whichis contradictory to its various heteroplasms in almost all otherchondroitin sulfotransferases (see Xu, D. et al., J. Biol. Chem., Mar.16, 2007; 282 (11): 8356-67). Along with complicated functions that areverified in diverse manners (see Bao, X. et al., (2005), J. Biol. Chem.,280, 23184-23193), CS-2OST carries a sulfo group to the 2-OH position ofhexauronic acid adjacent to a N-acetylated galactosamine residuecarrying 6-0 or 4-0 sulfo group (see Kobayashi, M. et al., (1999), J.Biol. Chem., 274, 10474-10480; Ohtake, S. et al., (2005), J. Biol.Chem., 280, 39115-39123).

Interestingly enough, the clear superimposing image of a decreasedCS-2OST mRNA expression, as the last step of CS variation in addition tothe absence of UA2S subcluster in CS under preeclampsia, reflects apoor, final tuning of CS that occurs commonly in the placenta of apregnant woman.

No one has yet discovered like the inventors that there is decrease inthe expression of sulfotransferases under preeclampsia. What is new andinteresting is that the decrease in the mRNA expression of CS-2OSTenzyme in the placenta of the pregnant woman with preeclampsia is inparallel with complete absence of its byproduct, UA2S.

The use of such data makes it possible to develop a predictive biomarkerkit through which the risk of preeclampsia in a pregnant woman can bepredicted.

To this end, the inventors obtained placental samples from normalpregnant women and from patients who are epidemiologically diagnosedwith preeclampsia. Those samples were ground, fats were removedtherefrom, proteins were hydrolyzed, and various GAGs were isolated byusing a column chromatography. These isolated GAGs were quantified bycarbazole assay (Blyscan assay) (refer to Table 1). In result, theaverage amount of GAGs (mg/g; dry sample) was lower in preeclampsiasamples than that of the control group.

In addition, for the analysis of a molecular weight and polydispersityof a sample, the inventors conducted PAGE, and computed an averagemolecular weight of GAGs based on the heparin oligosaccharide standard(refer to FIG. 1). In result, the average molecular weight of GAGs wasslightly lower in preeclampsia samples than that of the control group(refer to Table 2).

Then, the inventors depolymerized the GAG complex enzymatically, andconducted a disaccharide analysis using LC-MS (refer to FIG. 2). Indoing so, 8 kinds of disaccharides of Hep/HS were isolated (refer toFIG. 3).

A total of 12 samples were subjected to a compositional analysis of CSdisaccharides (refer to FIG. 2), wherein the compositional analysis wasmeasured in terms of Mean/SD (refer to Table 3). The comparison of CSdisaccharides between the preeclampsia sample group and the controlgroup shows that samples in the control group have more UA2S and TriSthan samples in the preeclampsia group (refer to Table 4). On the otherhand, Hp/HS disaccharides have N-sulfo disaccharides that are moreabundant in the control group than in the preeclampsia sample group.This implies that translocation of the N-sulfo group under preeclampsiaoccurs most frequently at the sixth carbon position. The preeclampsiasamples are very rich in tri-sulfo disaccharides (refer to Table 5).

The inventors also examined the variation in the amount of mRNA ofdiverse GAG synthesis regulatory enzymes by Quantitative Real-Time PCR(qRT-PCR). All primers were designed using gene-specific sequencesfostered by GenBank (refer to Table 6). The RT-PCR result confirms thatthe preeclampsia samples, compared with the control group placenta,showed noticeable decrease in the expression of diverse chondroitinsulfotransferases enzymes (refer to Table 7).

Hereinafter, the present invention will be explained in more detailthrough examples. However, it will be apparent to those skilled in theart that these examples are only for the purpose of explaining thepresent invention in detail, but not intended to limit the scope of theinvention.

Example 1 Acquisition—Purchase of Samples

Placental samples used for the present invention (e.g., placentalsamples from normal pregnant women and from patients who areepidemiologically diagnosed with preeclampsia) were provided by thedepartment of obstetrics and gynecology in Inje University Hospital.

Actinase E was purchased from Kaken Biochemicals (Tokyo, Japan), CS,chondroitin lyases and heparin lyases were purchased from Seikagaku(Tokyo, Japan), and polyacrylamide, urea, CHAPS, Alcian blue dye,2-cyanoacetamide and tetra-n-butylamonium hydrogen sulfate werepurchased from Sigma Chemical Company (St. Louis, Mo.). All othersamples were of reagent grade. Vivapure MAXI QH columns were purchasedfrom Viva science (Edgewood, N.J.).

Unsaturated disaccharide standards from CS (Di-0S ΔUA-Gal, Di-4SΔUA-Gal4S, Di-6S ΔUA-Gal6S, Di-UA2S ΔUA2S-Gal, Di-diS_(B) ΔUA2S-Gal4S,Di-diS_(D) ΔUA 2S-Gal6S, Di-diS_(E) ΔUA-Gal4S6S, Di-triS ΔUA2S-Gal4S6S)were purchased from Seikagaku Corporation (Japan), and chondroitinaseABC and ACII were also purchased from Seikagaku Corporation (Japan).

Unsaturated disaccharide standards from Hep/HS (Di-0S ΔUA-GlcNAc, Di-NSΔUA-GlcNS, Di-6S ΔUA-GlcNAc6S, Di-UA2S ΔUA2S-GlcNAc, Di-UA2SNSΔUA2S-GlcNS, Di-NS6S ΔUA-GlcNS6S, Di-UA2S6S ΔUA2S-GlcNAc6S, Di-triSΔUA2S-GlcNS6S) were purchased from Seikagaku Corporation (Japan).

A mixture (150 ng/μl) of disaccharide standards containing Di-0S, Di-4S,Di-6S, Di-diS_(D), Di-diS_(E), and Di-triS was indicated as std1. Amixture of disaccharide standards containing Di-0S, Di-4S, Di-UA2S,Di-diS_(D), Di-diS_(E), and Di-triS was indicated as std2. A mixture ofdisaccharide standards containing Di-0S, Di-4S, Di-6S, Di-diS_(B),Di-diS_(D), and Di-triS was indicated as std3. A mixture of disaccharidestandards containing Di-0S, Di-4S, Di-UA2S, Di-diS_(B), Di-diS_(E), andDi-triS was indicated as std4. A mixture of disaccharide standardscontaining Di-4S, Di-diS_(E), and double the amount of Di-6S andDi-diS_(D) was indicated as std5. A mixture of disaccharide standardscontaining Di-6S, Di-diS_(D), and double the amount of Di-4S andDi-diS_(E) was indicated as std6. A mixture of disaccharide standardscontaining Di-UA2S and double the amount of Di-6S was indicated as std7.

Example 2 Isolation and Purification of GAG

With the use of mortars and pestles, the samples were pulverizedtogether with dry ice to very fine particles of uniform size. Tissueswere rinsed with a mix solution of chloroform/methanol (2:1, 1:1, and1:2) (v/v), respectively, overnight, and thus defatted.

In 5 ml water, the fat-free samples were treated with 1% Actinase E (20mg/ml) at 55° C. for 18 hours to hydrolyze protein. Following thehydrolysis of protein, dry urea and dry CHAPS (2 wt % CHAPS and 8 Murea) were added to each of the samples. The obtained blurring solutionspassed through a syringe filter having a 0.2 μm film, and thus becametransparent. Vivapure MAXI QH spin columns reached the equilibrium statewith the addition of 3 ml of 8 M urea (pH 8.3) containing 2% CHAPS. Theclear, filtered samples were loaded on the Vivapure MAXI QH spin columnsin a centrifugal separator (500×g) and then perfused. First, the columnswere rinsed with 3 ml of 8 M urea (pH 8.3) containing 2% CHAPS at pH8.3. Then, they were rinsed five times with 5 ml of 200 mM NaCl.Further, the samples were rinsed three times with 1 ml of 16% NaCl toemit GAG from the spin column. To prepare an 80 vol % solution, 12 ml ofmethanol was added to each sample, and the mix solutions were allowed toreach the equilibrium state over the period of 18 hours at 4° C. Theobtained precipitates were centrifuged at 2500×g for 15 min andcollected. The precipitates were dissolved in 0.5 ml water andcollected. The collected precipitates were put in a freezer foradditional analysis on the collected GAGs.

Example 3 Quantification of GAGs by Carbazole Assay (Blyscan Assay)

With the isolated GAGs as a marker dye, the inventors conducted Blyscanassay using 1,9-dimethylmethylene blue (DMB) and quantified the amountof GAG in each of the samples by using standard CS.

As shown in Table 1 below, the inventors turned out that the averageamount of GAGs (mg/g; dry samples) in the control group was 2.78±0.48mg/g, and the average amount of GAGs in the preeclampsia samples was2.26±0.21 mg/g.

TABLE 1 Dry Description on Original mass GAG GAG No. sample mass (g) (g)(mg) (mg)/g MW (KD) 1 Normal control 0.9729 0.1094 0.205 1.87 8.4 groupA, Sample 1 2 Normal control 1.0842 0.1136 0.305 2.68 8.7 group A,Sample 2 3 Normal control 0.78 0.0817 0.226 2.77 1.3 group B, Sample 1 4Normal control 0.73 0.081 0.283 3.49 10.0 group B, Sample 2 5 Normalcontrol 0.769 0.0694 0.197 2.84 10.0 group C, Sample 1 6 Normal control0.545 0.063 0.190 3.0 10.0 group C, Sample 2 7 Preeclampsia 1.26 0.1090.211 1.94 9.3 patient A, Sample 1 8 Preeclampsia 1.1 0.099 0.233 2.359.7 patient A, Sample 2 9 Preeclampsia 0.795 0.094 0.200 2.13 9.6patient B, Sample 1 10 Preeclampsia 0.93 0.109 0.229 2.1 9.7 patient B,Sample 2 11 Preeclampsia 1.18 0.116 0.287 2.47 9.3 patient C, Sample 112 Preeclampsia 1.05 0.116 0.295 2.54 9.3 patient C, Sample 2

Example 4 PAGE Assay

For the analysis of a molecular weight and polydispersity of eachsample, PAGE assay was conducted. Electrophoresis of about 5 mg of GAGisolated to each lane was performed on the enzymatically preparedheparin oligosaccharide standard from bovine lung heparin. Then, gelvisualized by Alcian blue was digitized with UN-Scan-it software (SilkScientific, Utah, US), and the average molecular weight of GAGs wascomputed based on the heparin oligosaccharide standard (refer to FIG.1).

In result, as described in Table 2, an average molecular weight of GAGsfrom the control group and an average molecular weight of GAGs from thepreeclampsia samples were 9.57±0.73 kD and 9.48±0.19 kD, respectively.

Example 5 Disaccharide Assay Using LC-MS

<5-1> Enzymatic De-Polymerization of Complex GAG

The purified GAGs (20 μg/μl) were decomposed additionally bychondroitinase ABC 10U and ACII (SIGMA) and dissolved in 500 μl of 0.1%BSA, respectively. 5 μl of chondroitinase ABC and 5 μl of ACII wereadded, as enzyme solutions, to 20 μl of a substrate and allowed for thereaction overnight at 37° C. The products were then filtered by acentrifugal filter device (3000 Da cutoff, Millipore Corporation) tothus yield CS disaccharides. An accurately measured 100 μl of dd H₂O wasadded, and thereafter, the disaccharides were freeze-dried. Then,heparinase I, II, and III (Sigma) were added to the residual and allowedfor the reaction overnight at 37° C. Also, the products were filtered bythe centrifugal filter device (3000 Da cutoff, Millipore Corporation) tothereby yield Hep/HS disaccharides.

<5-2> LC-MS Assay

The LC-MS assay was conducted through LC-MS system (Agilent LC/MSD trapMS). Solutions A and B for HPLC were 15% and 70%, respectively, eachcontaining 37.5 mM NH₄CO₃ and 11.25 mM Tributylamine at the sameconcentration. The pH values of the solutions were adjusted to 6.5 withacetic acid. A flow rate was 10 μl/min. Separation was conducted using aC-18 column (Agilent) for 20 min for the solution A, followed by 20-45min for the solution B with the slope of a straight line from 0% to 50%.Column effluent returned to the source of EMI-MS to continue monitoring.

In result, in standard chromatography (refer to the top panel in FIG.2), Di-0S appeared at 5.3-5.4 min. Disaccharides having a singlesulfate, such as Di-UA2S, Di-4S and Di-6S, appeared at 10.3-12.5 min.Disulfated disaccharides like Di-diS_(B), Di-diS_(D) and Di-diS_(E)appeared at 10.3-12.5 min. Trisulfated disaccharide Di-triS appearedlastly at about 43.7 min. According to standard chromatograph of anothermix solution, Di-4S appeared at 11 min, Di-6S appeared at 10.3 min, andthe peak at the center was Di-UA2S. Di-diS_(E) appeared at about 38.6min, and Di-diS_(B) and Di-diS_(D) appeared together at 39.5 min.

FIG. 3 shows 8 kinds of disaccharides well isolated from Hep/HS. Allfractions were confirmed by MS (where data is not shown).

It is evident from the comparison on CS disaccharides between the samplegroup and the control group that the control group samples are moreabundant in UA2S and TriS than the preeclampsia samples (refer to Table4). In addition, it was verified that Hp/HS disaccharides have N-sulfodisaccharides that are more abundant in the control group compared withthe preeclampsia sample group. This result supports that translocationof the N-sulfo group under preeclampsia occurs most frequently at thesixth carbon position. Interestingly enough, as shown in Table 5, one ofthe preeclampsia samples had a very large amount of tri-sulfodisaccharide.

Tables 2 to 5 below show compositional analysis of CS disaccharides onall 12 samples, compositional analysis of CS disaccharides in terms ofMean/SD, compositional analysis of Hp/HS disaccharides on all 12samples, and compositional analysis of Hp/HS disaccharides in terms ofMean/SD, respectively.

TABLE 2 diS_(D) 0S 6S UA2S 4S diS_(E) or diS_(D) triS TPA Control group1 n.d 42.6% n.d 57.4% n.d n.d n.d 1715 2 n.d   16% 39.3% 42.7% n.d n.d  2% 24637 Mean n.d 29.3% 19.7% 50.1% n.d n.d   1% 13176 3 1.3%  4.1% 8.8% 83.7% n.d n.d 2.1% 16627 4 n.d 37.2% n.d 62.1% n.d n.d 0.7% 32478Mean 0.7% 20.7%  4.4% 72.9% n.d n.d 1.4% 24552 5 2.9% 24.4% n.d 66.5%n.d n.d 6.1% 5926 6 n.d 46.2% n.d 46.8% n.d n.d   7% 5443 Mean 1.5%35.3% n.d 56.7% n.d n.d 6.5% 5684 Preeclampsia 7 n.d 44.7% n.d 54.7% n.dn.d 0.6% 68532 8 n.d 49.2% n.d 50.8% n.d n.d 0.2% 169810 Mean n.d   47%n.d 52.7% n.d n.d 0.4% 119171 9 n.d 32.2% n.d 66.5% n.d n.d 1.2% 3055610  0.3%   37% n.d 62.4% n.d n.d 0.2% 101902 Mean 0.15%  34.6% n.d 64.5%n.d n.d 0.7% 66229 11  n.d 43.3% n.d 56.7% n.d n.d n.d 236590 12  n.d42.4% n.d 57.6% n.d n.d n.d 187563 Mean n.d 42.9% n.d 57.1% n.d n.d n.d212076

TABLE 3 diS_(D) or 0S 6S UA2S 4S diS_(E) diS_(D) triS TPA Control Mean0.7% 28.4%   8% 59.9% n.d n.d 2.98%  14471 group ±SD 0.7% 0.7% 10.3%11.8% 0 0 3.1% Preeclampsia Mean n.d 41.5% n.d 58.1% n.d n.d 0.3% 132492±SD 0.1% 6.3% 0  5.9% 0 0 0.3%

TABLE 4 0S NS 6S UA2S UA2SNS NS6S UA2S6S triS TPA Control group 1 n.d63.6% 36.4% n.d n.d n.d n.d n.d 11579 2 n.d 74.3% 25.7% n.d n.d n.d n.dn.d 5114 Mean n.d 69.0% 31.0% n.d n.d n.d n.d n.d 8346 3 n.d 48.2% 51.8%n.d n.d n.d n.d n.d 25163 4 n.d 34.7% 60.7% n.d 0.4% n.d n.d 4.3% 54561Mean n.d 41.5% 56.2% n.d 0.2% n.d n.d 2.1% 39862 5 n.d 69.0% 26.0% 5.0%n.d n.d n.d n.d 17876 6 n.d 75.5% 24.5% n.d n.d n.d n.d n.d 9447 Meann.d 72.3% 25.2% 2.5% n.d n.d n.d n.d 13661 Preeclampsia 7 n.d 49.0%51.0% n.d n.d n.d n.d n.d 45024 8 n.d 28.8% 50.9% 20.3%  n.d n.d n.d n.d104083 Mean n.d 38.9% 51.0% 10.1%  n.d n.d n.d n.d 74553 9 n.d 12.4%84.6% n.d 3.0% n.d n.d n.d 3586 10  n.d 16.9% 78.2% n.d 1.9% n.d n.d3.0% 11064 Mean n.d 14.7% 81.4% n.d 2.5% n.d n.d 0.1% 7325 11  n.d 10.8%69.9% n.d 1.2% n.d n.d 18.1%  43001 12  n.d 12.6% 16.7% 9.1% 8.4% n.dn.d 53.1%  3740 Mean n.d 11.7% 43.3% 4.6% 4.8% n.d n.d 35.6%  23374

TABLE 5 0S NS 6S UA2S UA2SNS NS6S UA2S6S triS TPA Control Mean n.d 60.8%37.5% 0.8% n.d n.d n.d n.d 20623 group ±SD 0 16.9% 16.5% 1.4% 0.1% 0 0 0Preeclampsia Mean n.d 21.8% 58.6% 4.9% 2.4% n.d n.d 12.4% 35083 ±SD 010.5% 14.2% 3.6% 1.7% 0 0 14.2%

Example 6 qRT-PCR

Variation in the amount of mRNA of diverse GAG synthesis regulatoryenzymes was examined by qRT-PCR. Total RNA amount was extracted, usingan RNA Ambion RiboPure total RNA isolation kit, frompreeclampsia-infected placentas and normal placentas. Following thetreatment with deoxyribonuclease I (Takara Holdings Inc., Japan), totalRNA was reversely transcribed by SuperScript II reverse transcriptase(Invitrogen, Carlsbad, Calif.). Next, qRT-PCR was conducted usingiCycler iQ system (Bio-Rad, Hercules, Calif.). Primers were designedusing software Primer 3 (developed by Steve Rozen and Helen J.Skaletsky). All primers were designed using gene-specific sequencesfostered by GenBank. The gene expression data was normalized to GAPDH asmuch as house keeping gene used as an internal standard for research.Primer sequences are presented in Table 6 (forward primers and reverseprimers used for qRT-PCR) as follows.

TABLE 6 GenBank Primer Sequence Accession Forward Reverse Predicted #direction direction size (bp) (GAPDH) NM_002046.3 5′ACCACAGTCCA5′TCCACCACCTG 452 TGCCATCAC3′ TTGCTGTA3′ Sequence No. 1 Sequence No. 2Chondroitin NM_018413 5′GTGGGGAGAGG 5′ACAGACAAGAA 200 4-0- GAGAGAATC3′CGACCCATC3′ sulfo- Sequence No. 3 Sequence No. 4 transferase (C4ST)Chondroitin NM_004273 5′CCCAAAGTCAG 5′ACAAGCAAACC 189 6- AAAGCGAAG3′CACCAACTC3′ sulfo- Sequence No. 5 Sequence No. 6 transferase (C6S)Dermatan/ NM_005715 5′TCTGAGCCTGA 5′CACCTGCACAG 153 chondroitinCCACACAGATG3′ AACTCAGGA3′ sulfate Sequence No. 7 Sequence No. 8 2-sulfo-transferase (CS20ST) Heparan NM_001543 5′CCCAGTGGCCC 5′GTCCATCACTT 205N-deacetlace/ TAAAGTACA3′ TGGCAGGTT3′ N-Sulfo- Sequence No. 9 SequenceNo. 10 transferase-1 (NDST-1) Heparan NM_004807 5′GTACAACCTGG5′CGCGTGCTATT 243 sulfate CCAACAACC3′ GTACTGCAT3′ 6-O-sulfo- SequenceNo. 11 Sequence No. 12 transferase 1 (HS6S)

It was verified from the RT-PCR result that the preeclampsia samples,compared with the control group placenta, showed noticeable decrease inthe expression of diverse chondroitin sulfotransferases enzymes (referto Table 7). Although the expression of heparan sulfate6-O-sulfotransferase in preeclampsia was markedly decreased, NDST-1 mRNAexpression showed no significant change even in preeclampsia.

TABLE 7 Quantitative analysis of relative change in mRNA expression ofdiverse GAG synthesis enzymes based on qRT-PCR of placenta of women withpreeclampsia Relative expression^(d) Gene C_(T) ^(a) ΔC_(T) ^(b) ΔΔC_(T)^(c) to control group chondroitin 4-O-sulfotransferase 1 (C4ST)Preeclampsia GAPDH 16.54 ± 0.38 5.25 ± 0.61 1.35 0.39 C4ST 21.79 ± 0.24Control GAPDH 17.43 ± 0.17  3.9 ± 0.25 group C4ST 21.33 ± 0.18chondroitin 6-sulfotransferase (C6S) Preeclampsia GAPDH 16.54 ± 0.387.913 ± 0.45  2.9 0.134 C6S 24.45 ± 0.61 Control GAPDH 17.43 ± 0.17   5± 0.6 group C6S 22.43 ± 0.43 dermatan/chondroitin sulfate2-sulfotransferase (CS-2OST) Preeclampsia GAPDH 16.54 ± 0.38 8.45 ± 0.642.3 0.2 CS2S 24.99 ± 0.49 Control GAPDH 17.43 ± 0.17 6.15 ± 0.70 groupCS2S 23.58 ± 0.53 heparan N-deacetylase/N-sulfotransferase-1 (NDST-1)Preeclampsia GAPDH 16.54 ± 0.38 3.85 ± 0.29 −0.167 1.123 HDS-1 20.53 ±0.48 Control GAPDH 17.43 ± 0.17 4.02 ± 1.03 group HDS-1 21.45 ± 0.86heparan sulfate 6-O-sulfotransferase 1 (HS6ST) Preeclampsia GAPDH 16.54± 0.38 7.21 ± 1.29 1.78 0.29 HS6S 23.75 ± 1.12 Control GAPDH 17.43 ±0.17 5.43 ±± .02 group HS6S 22.87 ± 0.85

where a: C_(T) data average for each sample,

b: ΔC_(T) value is calculated by subtracting GAPDH C₁ from each sampleC_(T),

c: ΔΔC_(T) value is calculated by subtracting control group ΔC_(T) fromeach preeclampsia sample ΔC_(T), and

d: relative expression to the control group is calculated using equation2^(−ΔΔCT).

As discussed earlier, the biomarker and composition for diagnosis ofpreeclampsia according to the present invention are not only effectivein the diagnosis of preeclampsia, but also useful for developing apredictive biomarker kit through which the risk of preeclampsia in apregnant woman can be predicted.

While the invention has been shown and described with respect to thepreferred embodiments, it will be understood by those skilled in the artthat various changes and modifications may be made without departingfrom the spirit and the scope of the invention as defined in thefollowing claims.

1. A biomarker for diagnosis of preeclampsia using an enzyme selectedfrom the group consisting of placental chondroitin 4-O-sulfotransferase1 (C4ST), chondroitin 6-sulfotransferase (C6S), heparan sulfate6-O-sulfotransferase 1 (HS6S), and dermatan/chondroitin sulfate2-sulfotransferase (CS-2OST), or uronic acid-2-sulfate (UA2S).
 2. Thebiomarker of claim 1, wherein the biomarker is dermatan/chondroitinsulfate 2-sulfotransferase (CS-2OST) or uronic acid-2-sulfate (UA2S). 3.The biomarker of claim 1, wherein chondroitin 4-O-sulfotransferase 1(C4ST), chondroitin 6-sulfotransferase (C6S), heparan sulfate6-O-sulfotransferase 1 (HS6S) and dermatan/chondroitin sulfate2-sulfotransferase (CS-2OST) have NCBI GenBank Accession Numbers ofNM_(—)018413, NM_(—)004273, NM_(—)004807 and NM_(—)005715, respectively.4. A composition for diagnosis of preeclampsia, comprising one or moreprimer pairs selected from the group consisting of primer pairs with thesequences of: (Primer Pair No. 1) Forward: 5′GTGGGGAGAGGGAGAGAATC3′(Sequence No. 1), Reverse: 5′ACAGACAAGAACGACCCATC3′ (Sequence No. 2);(Primer Pair No. 2) Forward: 5′CCCAAAGTCAGAAAGCGAAG3′ (Sequence No. 3),Reverse: 5′ACAAGCAAACCCACCAACTC3′ (Sequence No. 4); (Primer Pair No. 3)Forward: 5′TCTGAGCCTGACCACAGATG3′ (Sequence No. 5), Reverse:5′CACCTGCACAGAACTCAGGA3′ (Sequence No. 6); (Primer Pair No. 4) Forward:5′CCCAGTGGCCCTAAAGTACA3′ (Sequence No. 7), Reverse:5′GTCCATCACTTTGGCAGGTT3′ (Sequence No. 8); and (Primer Pair No. 5)Forward: 5′GTACAACCTGGCCAACAACC3′ (Sequence No. 9), Reverse:5″CGCGTGCTATTGTACTGCAT3′ (Sequence No. 10).